Method for producing lactone

ABSTRACT

This invention relates to a method for producing a lactone comprising culturing  Candida sorbophila  in a medium containing at least one selected from the group consisting of a hydroxy fatty acid, a hydroxy fatty acid derivative, and a hydrolysate of a hydroxy fatty acid derivative and recovering the produced lactone from the medium. This invention also relates to a method for producing a lactone comprising culturing  Candida sorbophila  in a medium containing at least one selected from the group consisting of a hydroxy fatty acid, a hydroxy fatty acid derivative, and a hydrolysate of a hydroxy fatty acid derivative and lactonizing the lactone precursor hydroxy fatty acid produced in the medium.

TECHNICAL FIELD

The present invention relates to a method for producing a lactone, whichis useful for flavor and fragrance substances, pharmaceuticalintermediates, and the like, with the use of microorganisms.

BACKGROUND ART

Aromatic substances are roughly classified into two categories: that is,chemically synthesized (so-called “synthetic aromatics”); andnon-chemically synthesized (so-called “natural aromatics”), depending onthe starting material thereof or the method for producing the same.Recently, consumers tend to prefer “natural products” over “syntheticproducts.” However, natural substances contain only very small amountsof, for example, optically active substances of γ-decalactone(R-γ-decalactone or S-γ-decalactone) and δ-decalactone, which areimportant ingredients of natural food flavor. Thus, processes such asextraction of such optically active substances with high optical purityor isolation thereof via other means are disadvantageous from technicaland economical viewpoints. As a result, large quantities of syntheticproducts are generally supplied at low prices at present. In contrast,the scale of manufacturing natural products is small, and such naturalproducts are often expensive.

Development of a method for supplying large quantities of theaforementioned natural aromatics at prices as low as those of currentlyused synthetic aromatics has been awaited. Among the methods forsupplying large quantities of natural aromatics that have been proposeda microorganism-based fermentation technique has drawn attention. Inthis technique, natural R-γ-decalactone is produced from naturalmaterials or degradation products thereof via biological or physicaltechniques without any chemical techniques.

For example, JP Patent Publication (Kokai) No. 59-82090 A (1984)discloses a microorganism-based method for producing γ-decalactone fromcastor oil or a hydrolysate thereof. In this method, microorganisms suchas Aspergillus oryzae, Candida rugosa, Geotrichum klebannii, andYarrowia lipolytica are used to produce γ-hydroxydecanoic acid, and theresultant is acidified with the addition of hydrochloric acid or thelike, followed by heating for lactonization, and thus γ-decalactone isproduced. JP Patent Publication (Kokai) No.63-56295 A (1988) and thereport by K. A. Maume et al. (Biocatalysis, vol. 5, 79–97, 1991)disclose a method for producing γ-decalactone wherein γ-hydroxydecanoicacid is produced from ricinoleic acid sources using Sporobolomycesodorus or Rhodotorula glutinis, and the resultant is also lactonized. JPPatent Publication No. 2-174685 A (1990) discloses a method forproducing γ-decalactone from castor oil or ricinoleic acid viaproduction of γ-hydroxydecanoic acid with the use of microorganisms suchas Aspergillus niger. JP Patent Publication No. 3-117494 A (1991)discloses the same technique with the use of microorganisms such asSaccharomyces cerevisiae. Prior to the disclosure of these techniques,S. Okui et al. reported the presence of γ-hydroxydecanoic acid andγ-decalactone as intermediates during the process for oxidizing anddegrading ricinoleic acid with the use of several cell strains of thegenus Candida (J. Biochem., vol. 54, No. 6, 536–540, 1963). Also, EP997533 discloses a method for producing γ-decalactone from castor oil at12 g per liter using Yarrowia lipolytica. This method for production,however, is extremely disadvantageous from the viewpoint of productionefficiency due to the necessity of the use of an emulsifier or pHadjuster during the culture and a small amount of a starting material,i.e., castor oil, to be added to the culture system, which is as low as0.0247 kg/L. Further, the microorganisms disclosed in such publicationsand the like, which are of species different from those of themicroorganisms used in the present invention, are not always suitablefor practical use because of the difficulty of separation of cellstrains from culture products and insufficient amounts of productionfrom an economical viewpoint.

Alternatively, a method for producing γ-decalactone from a sugarsubstrate with the aid of Sporobolomyces odorus (S. Taqhara et al.,Agric. Biol. Chem., vol. 36, No. 13, 2585–2587, 1972; N. Jourdain etal., “Top. Flavour Res., Proc. Int. Conf,” H. Eichhorn, 427–441, 1985)or Fusarium poae (J. Sarris et al., Agric. Biol. chem., vol. 49, No. 11,3227–3230, 1985), which is an example of methods in which a componentother than castor oil or a hydrolysate thereof is employed as a carbonsource, has been reported. However, these techniques are not suitablefor industrial-scale production since only a very small amount ofγ-decalactone is produced.

Accordingly, development of a method for effectively producingγ-hydroxydecanoic acid and γ-decalactone that does not require the useof an emulsifier or pH adjuster and that allows the addition of a highlyconcentrated starting material, i.e., castor oil and/or a hydrolysatethereof, has been awaited.

It is also reported that the abundance of R-γ-decalactone enantiomer innaturally occurring γ-decalactone is excessive (A. Bernreuther et al.,J. Chromatography, 481, 363, 1989). A method for producing a pureoptically active form of such R-γ-decalactone via chemical synthesis isdisclosed in JP Patent Publication No. 4-108782 A (1992).

R-γ-decalactone can also be produced by selectively separatingR-γ-decalactone from racemic mixtures as extracted from naturalsubstances by a technique known to a person skilled in the art. Becauseof the very small amount of R-γ-decalactone in natural substances and aphysical difficulty in separating R-γ-decalactone from other volatilecompounds, however, extraction thereof from natural substances is notcost-effective. In order to deal with increasing demands for naturalcompounds as mentioned above, development of a method for effectivelyproducing natural R-γ-decalactone using techniques different from thechemical synthesis thereof or the separation thereof from racemicmixtures, has been awaited.

A method for producing δ-decalactone using microorganisms, which makesuse of the reducing ability of fungi, particularly that of yeast, hasbeen proposed. For example. JP Patent Publication No. 3-155792 A (1991)discloses a method for producing 5-decanolide from naturally occurring2-decen-1,5-olide with the utilization of the reducing ability ofSaccharomyces cerevisiae. JP Patent Publication No. 6-225781 A (1994)reports a method for producing δ-decanolide, δ-dodecanolide, or amixture thereof from a substrate material containing a correspondingunsaturated lactone, i.e., δ-decen-2-olide, δ-dodecen-2-olide, or amixture thereof, via biohydrogenation with the use of yeast such asSaccharomyces delbrueckii. However, a technique of chemical conversionutilizing the reducing ability of yeast is still problematic in termsof, for example, the difficulty in acting on high concentrations of thesubstrate and the necessity of a long period of time to obtain thesubstance of interest.

DISCLOSURE OF THE INVENTION

This application claims the priority of Japanese Patent Application No.2002-190616, the disclosure of which is incorporated herein.

The present invention has been made in order to overcome the problemsmentioned above. It is an object of the present invention to provide amethod for effectively producing a natural lactone including anoptically active lactone such as an optically active γ-decalactone andan optically active δ-decalactone, with the use of microorganisms.

In order to attain the above object, the present inventors have searchedfor microorganisms extensively from existing cell strains and in naturethat can accumulate highly concentrated γ-hydroxydecanoic acid in aculture medium with castor oil and/or a hydrolysate thereof as a carbonsource. As a result, they have found that use of Candida sorbophilaenabled production and accumulation of γ-hydroxydecanoic acid and/or anoptically active γ-decalactone in a culture medium with high efficiencywhile using no emulsifier or pH adjuster and adding at least onestarting material selected from the group consisting of castor oil, acastor oil hydrolysate, ricinoleic acid, and lesquerolic acid at highconcentration. Further, they have also found that optically activeγ-decalactone could be easily produced by heating the obtainedγ-hydroxydecanoic acid under acidic conditions. The thus producedoptically active γ-decalactone was recovered with very highproductivity. Furthermore, they have also found that the aforementionedCandida sorbophila enabled the production of a variety of opticallyactive lactones by employing a variety of hydroxy fatty acids as carbonsources.

The present invention has been completed based on such findings and isas described below.

(1) A method for producing a lactone comprising culturing Candidasorbophila in a medium containing at least one selected from the groupconsisting of a hydroxy fatty acid, a hydroxy fatty acid derivative, anda hydrolysate of a hydroxy fatty acid derivative, and recovering theproduced lactone from the medium.

(2) A method for producing a lactone comprising culturing Candidasorbophila in a medium containing at least one selected from the groupconsisting of a hydroxy fatty acid, a hydroxy fatty acid derivative, anda hydrolysate of a hydroxy fatty acid derivative and lactonizing alactone precursor hydroxy fatty acid produced in the medium.

(3) The method according to (1) or (2), wherein the Candida sorbophilais at least one selected from the group consisting of the Candidasorbophila strain ATCC 74362, the Candida sorbophila strain ATCC 60130,the Candida sorbophila strain IFO 1583, and the Candida sorbophilastrain FC 58 deposited under the accession number FERM BP-8388.

(4) The method according to (1) or (2), wherein the lactone isrepresented by the general formula (1):

wherein ring A represents a lactone ring; R¹ represents a hydrogen atom,a hydrocarbon group, a substituted hydrocarbon group, a heterocyclicgroup, or a substituted heterocyclic group; and R² represents a hydrogenatom a hydrocarbon group or a substituted hydrocarbon group; in whichring A and R² may be bonded to form a ring.

(5) The method according to (1) or (2), wherein the lactone is anoptically active lactone.

(6) The method according to (1) or (2), wherein the hydroxy fatty acidis represented by the general formula (2):

wherein R¹ represents a hydrogen atom, a hydrocarbon group, asubstituted hydrocarbon group, a heterocyclic group, or a substitutedheterocyclic group; R² represents a hydrogen atom, a hydrocarbon group,or a substituted hydrocarbon group; and R³ represents an optionallysubstituted divalent hydrocarbon group having a 4 or more-carbon chain;in which R² and R³ may be bonded to form a ring.

(7) The method according to (1) or (2), wherein the hydroxy fatty acidderivative is an alkyl ester of hydroxy fatty acid or a glyceride ofhydroxy fatty acid.

(8) The method according to (7), wherein the alkyl ester of hydroxyfatty acid is represented by the general formula (3):

wherein R¹ represents a hydrogen atom, a hydrocarbon group, asubstituted hydrocarbon group, a heterocyclic group, or a substitutedheterocyclic group; R² represents a hydrogen atom, a hydrocarbon group,or a substituted hydrocarbon group; R³ represents an optionallysubstituted divalent hydrocarbon group having a 4 or more-carbon chain;and R⁴ represents an alkyl group; in which R² and R³ may be bonded loform a ring.

(9) The method according to (7), wherein the glyceride of hydroxy fattyacid is represented by the general formula (4):

wherein R⁶ to R⁸ each independently represents a hydrogen atom or agroup represented by the general formula (6):

wherein R¹ represents a hydrogen atom, a hydrocarbon group, asubstituted hydrocarbon group, a heterocyclic group, or a substitutedheterocyclic group; R² represents a hydrogen atom, a hydrocarbon group,or a substituted hydrocarbon group; R³ represents an optionallysubstituted divalent hydrocarbon group having a 4 or more-carbon chain;and R⁴ represents an alkyl group; in which R² and R³ may be bonded toform a ring, provided that at least one of R⁶ to R⁸ is a grouprepresented by the above general formula (6).

(10) The method according to (1) or (2), wherein Candida sorbophila iscultured in a medium containing at least one selected from the groupconsisting of castor oil, a castor oil hydrolysate, ricinoleic acid,11-hydroxypalmitic acid, lesquerolic acid, 10-hydroxystearic acid,10-hydroxypalmitic acid, and ethyl 11-hydroxypalmitate.

(11) The method according to (2), wherein the lactone precursor hydroxyfatty acid is a hydroxy fatty acid of 4 or more carbon atoms having ahydroxy group at position 4 or 5 thereof.

(12) The method according to (1) or (2), wherein the lactone is any oneselected from the group consisting of γ-decalactone, γ-valerolactone,γ-hexalactone, γ-heptalactone, γ-octalactone, γ-nonalactone,γ-undecalactone, γ-dodecalactone, γ-tridecalactone, γ-tetradecalactone,δ-decalactone, δ-hexalactone, δ-heptalactone, δ-octalactone,δ-nonalactone, δ-undecalactone, δ-dodecalactone, δ-tridecalactone, andδ-tetradecalactone.

(13) A method for producing a lactone precursor hydroxy fatty acidcomprising culturing Candida sorbophila in a medium containing at leastone selected from the group consisting of a hydroxy fatty acid, ahydroxy fatty acid derivative, and a hydrolysate of a hydroxy fatty acidderivative.

(14) A method for producing γ-decalactone comprising culturing Candidasorbophila in a medium containing at least one selected from the groupconsisting of castor oil, a castor oil hydrolysate, ricinoleic acid, andlesquerolic acid, and recovering the produced γ-decalactone from themedium.

(15) A method for producing γ-decalactone comprising culturing Candidasorbophila in a medium containing at least one selected from the groupconsisting of castor oil, a castor oil hydrolysate, ricinoleic acid, andlesquerolic acid, and lactonizing γ-hydroxydecanoic acid produced in themedium.

(16) The method according to (14) or (15), wherein γ-decalactone is anoptically active γ-decalactone.

(17) The method according to (14) or (15), wherein at least one selectedfrom the group consisting of castor oil, a castor oil hydrolysate,ricinoleic acid, and lesquerolic acid is castor oil and/or a castor oilhydrolysate.

(18) A method for producing δ-decalactone comprising culturing Candidasorbophila in a medium containing 11-hydroxypalmitic acid and/or ethyl11-hydroxypalmitate and recovering the produced δ-decalactone from themedium.

(19) A method for producing δ-decalactone comprising culturing Candidasorbophila in a medium containing 11-hydroxypalmitic acid and/or ethyl11-hydroxypalmitate and lactonizing δ-hydroxydecanoic acid produced inthe medium.

(20) The method according to (18) or (19), wherein δ-decalactone is anoptically active δ-decalactone.

(21) The method according to (14), (15), (18), or (19), wherein theCandida sorbophila is at least one selected from the group consisting ofthe Candida sorbophila strain ATCC 74362, the Candida sorbophila strainATCC 60130, the Candida sorbophila strain IFO 1583, and the Candidasorbophila strain FC 58 deposited under the accession number FERMBP-8388.

(22) Use of Candida sorbophila for producing a lactone.

(23) A Candida sorbophila strain FERM BP-8388.

The production method of a lactone according to the present invention iscarried out by culturing Candida sorbophila in a medium containing atleast one selected from the group consisting of a hydroxy fatty acid, ahydroxy fatty acid derivative, and a hydrolysate of a hydroxy fatty acidderivative to produce a lactone and then recovering the lactone from themedium.

The production method of a lactone according to the present invention isalso carried out by culturing Candida sorbophila in a medium containingat least one selected from the group consisting of a hydroxy fatty acid,a hydroxy fatty acid derivative, and a hydrolysate of a hydroxy fattyacid derivative to produce a lactone precursor hydroxy fatty acid andthen lactonizing the lactone precursor hydroxy fatty acid.

Hereafter, the method for producing a lactone according to the presentinvention is described in detail.

(1) Candida sorbophila

Specific examples of Candida sorbophila used in the present inventioninclude, but are not limited to, the Candida sorbophila strain FC 58,the Candida sorbophila strain ATCC 74362, the Candida sorbophila strainATCC 60130, and the Candida sorbophila strain IFO 1583. The Candidasorbophila strain FC 58 was deposited at the International PatentOrganism Depository of the National Institute of Advanced IndustrialScience and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan) as of Jun. 10, 2002 under the accession number: FERMBP-8388 (the original deposit). A request for transfer of the depositionfrom the original to the international deposition under the BudapestTreaty was accepted as of May 28, 2003.

The aforementioned Candida sorbophila strain FC 58 was separated fromcommon soil in Kanagawa prefecture, Japan, in accordance with aconventional technique, the mycological properties thereof wereidentified, and the identified properties were studied in accordancewith the taxonomic textbooks (Kurtzman, C. P. et al., “The Yeasts, ATaxonomic Study” 4th edition, 1998, Elsevier Science B. V.; and Barnett,J. A. et al., “Yeasts: Characteristics and identification” 3rd ed). As aresult, the Candida sorbophila strain FC 58 was identified to be amicroorganism of the genus Candida sorbophila. Thus, this microorganismseparated from nature was designated as the Candida sorbophila strain FC58 (hereafter abbreviated as the “FC 58 strain”).

The mycological properties of the FC 58 strain that can be preferablyused in the present invention are as follows.

(1) Growth in YM liquid medium: the form primarily changes fromspherical to oval after the strain has been cultured at 24° C. to 27° C.for 24 hours.

(2) Growth in YM agar medium: the color of the strain becomes betweenwhite and cream and the strain is moistened after it has been culturedat 24° C. to 27° C. for 2 to 3 days.

(3) Form: spherical to oval, proliferated by multipolar budding,formation of pseudomycelium and formation of ascospore when it iscultured in medium of Adams, Gorodokowa, malt, YM, V-8, or potatodextrose are not observed.

(4) Optimal growth conditions: at 24° C. to 27° C., pH 5.5 to 6.0.

(5) Maximal temperature for growth: at 35° C. to 37° C.

(6) Vitamin requirement: it cannot grow in a vitamin-deficient mediumBiotin, pyridoxine, or thiamine is required.

(7) Fermentation: glucose (−), galactose (−), sucrose (−), maltose (−),lactose (−), raffinose (−), trehalose (−).

(8) Assimilability: galactose (+), sorbose (+), sucrose (−) maltose (−),trehalose (−), lactose (−), raffinose (−), cellobiose (−), melibiose(−), melezitose (−), starch (−), D-xylose (−), L-arabinose (−),D-ribose. (−), D-rhamnose (−), D-glucosamine (−), N-acetyl-D-glucosamine(−), glycerol (weak), erythritol (−), ribitol (−), D-mannitol (+),lactate (weak), citrate (−), inositol (−)

The aforementioned “ATCC” and “IFO” are abbreviations of “American TypeCulture Collection” and “Institution for Fermentation, Osaka, Japan,”respectively. A numerical value provided with “ATCC” or “IFO” representsthe catalog number of each strain. The aforementioned Candida sorbophilarepresented by the numerical value following “ATCC” or “IFO” can beobtained from respective organizations based on those catalog numbers.

When the Candida sorbophila used in the present invention is cultured ina medium containing a hydroxy fatty acid, a hydroxy fatty acidderivative, and/or a hydrolysate of a hydroxy fatty acid derivative, itcan produce a lactone precursor hydroxy fatty acid by β-oxidation, andthen may produce a lactone by lactonizing the generated lactoneprecursor hydroxy fatty acid, resulting in accumulation of them in themedium. When the Candida sorbophila used in the present invention iscultured in a medium containing a hydroxy fatty acid derivative underadequate culture conditions, this strain hydrolyzes the hydroxy fattyacid derivative and then β-oxidizes the hydrolysate to produce a lactoneprecursor hydroxy fatty acid. The product is then accumulated in themedium. In such a case, lactonization of the lactone precursor hydroxyfatty acid that is produced and accumulated in the medium can result inthe generation of a desired lactone.

When the Candida sorbophila used in the present invention is cultured ina medium containing, for example, castor oil, it can hydrolyze thecastor oil. Further, during the culture, γ-hydroxydecanoic acid and/orγ-decalactone can be produced and accumulated in the medium byβ-oxidation of a hydrolysate of such castor oil.

(2) Medium for Producing a Lactone

In the present invention, a medium that contains at least one member, asa carbon source, selected from the group consisting of a hydroxy fattyacid, a hydroxy fatty acid derivative, and a hydrolysate of a hydroxyfatty acid derivative and in which Candida sorbophila can grow, is usedin the present method for producing a lactone.

The hydroxy fatty acid or hydrolysate of a hydroxy fatty acid derivativeused in the present invention is not particularly limited as long as itcan be β-oxidized by Candida sorbophila, thereby producing the lactoneprecursor hydroxy fatty acid. The hydroxy fatty acid derivative used inthe present invention is not particularly limited as long as it ishydrolyzed by Candida sorbophila and the lactone precursor hydroxy fattyacid can be produced from the resulting hydrolysate via β-oxidation.

The hydroxy fatty acid, which has 6 or more, preferably 6 to 25, andmore preferably 6 to 20 carbon atoms, and has a hydroxy group in atleast the 6-position, and preferably in the 6- to 20-positions from thecarbon atom in the carboxy group, is preferably used in the presentinvention.

A specific example of a hydroxy fatty acid, for example, is representedby the general formula (2):

wherein R¹ represents a hydrogen atom, a hydrocarbon group, asubstituted hydrocarbon group, a heterocyclic group, or a substitutedheterocyclic group; R² represents a hydrogen atom, a hydrocarbon group,or a substituted hydrocarbon group; R³ represents an optionallysubstituted divalent hydrocarbon group having a 4 or more-carbon chain;in which either R¹ and R² or R² and R³ may be bonded to form a ring.

These groups in the general formula (2) are described below.

Examples of the hydrocarbon group, for example, include alkyl, alkylhaving an unsaturated bond, aryl, aralkyl and the like.

Alkyl may be linear, branched, or cyclic. For example, alkyl has atleast one preferably 1 to 20, more preferably 1 to 15, and furtherpreferably 1 to 10 carbon atoms. Specific examples thereof includemethyl, ethyl, n-propyl, 2-propyl, n-butyl, 2-butyl, isobutyl,tert-butyl, n-pentyl, 2-pentyl, tert-pentyl, 2-methylbutyl,3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-hexyl, 3-hexyl,tert-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,2-methylpentan-3-yl, n-heptyl, n-octyl, n-nonyl, n-decyl, cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl.

The alkyl having an unsaturated bond includes alkyl having at least oneunsaturated bond such as a double bond and the like in its chain.Specific examples thereof include alkenyl, alkadienyl, alkatrienyl andthe like.

Alkenyl has one double bond in the chain of the aforementioned alkyl.For example, it may be linear, branched, or cyclic, and it may have atleast 2, preferably 2 to 20, more preferably 2 to 15, and furtherpreferably 2 to 10 carbon atoms. Specific examples thereof includeethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, nonanyl, decenyland the like.

Alkadienyl has two double bonds in the chain of the aforementionedalkyl. For example, it may be linear, branched, or cyclic, and it mayhave at least 4, preferably 4 to 20, more preferably 4 to 15, andfurther preferably 4 to 10 carbon atoms. Specific examples thereofinclude 1,3-butadienyl, 2,4-butadienyl, 2,3-dimethyl-1,3-butadienyl andthe like.

The aryl includes one that has 6 to 14 carbon atoms. Specific examplesthereof include phenyl, naphthyl, anthryl, biphenyl and the like.

The aralkyl includes a group derived from the aforementioned alkyl bysubstitution of at least one hydrogen atom with the aforementioned aryl.For example, aralkyl preferably has 7 to 12 carbon atoms. Specificexamples thereof include benzyl, 2-phenylethyl, 1-phenylpropyl,3-naphthylpropyl and the like.

Examples of the heterocyclic group include an aliphatic heterocyclicgroup and an aromatic heterocyclic group.

The aliphatic heterocyclic group includes, for example, a 5- to8-membered and preferably a 5- or 6-membered monocyclic, polycyclic, orfused-ring aliphatic heterocyclic group, which has 2 to 14 carbon atomsand contains as heteroatoms at least one and preferably 1 to 3heteroatoms, such as nitrogen, oxygen, and sulfur atoms. Specificexamples of the aliphatic heterocyclic group include, for example,pyrrolidyl-2-one, piperidino, piperazinyl, morpholino, tetrahydrofuryl,tetrahydropyranyl and the like.

The aromatic heterocyclic group, for example, includes a 5- to8-membered and preferably a 5- or 6-membered monocyclic, polycyclic, orfused-ring heteroaryl, which has 2 to 15 carbon atoms, and contains asheteroatoms at least one and preferably 1 to 3 heteroatoms, such as anitrogen, oxygen and sulfur atom. Specific examples thereof includefuryl, thienyl, pyridyl, pyrimidyl, pyrazyl, pyridazyl, pyrazolyl,imidazolyl, oxazolyl, thiazolyl, benzofuryl, benzothienyl, quinolyl,isoquinolyl, quinoxalyl, phthalazyl, quinazolyl, naphthylidyl, cinnolyl,benzimidazolyl, benzoxazolyl, benzothiazolyl and the like.

The substituted hydrocarbon group includes substituted alkyl,substituted alkyl having an unsaturated bond, substituted aryl, andsubstituted aralkyl, wherein at least one hydrogen atom in theaforementioned hydrocarbon group has been substituted with asubstituent.

The aforementioned substituent includes hydrocarbon group, substitutedhydrocarbon, heterocyclic group, heterocyclic group, alkoxy, aryloxy,aralkyloxy, alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, acyl,acyloxy, alkylthio, aralkylthio, arylthio, halogen, alkylenedioxy,amino, substituted amino, hydrazino, cyano, nitro, hydroxy, hydroxysubstituted with a protecting group, carboxy, sulfonylamino, sulfo,amide phosphate, substituted silyl and the like.

These groups represented by R¹ are preferably alkyl or alkyl having anunsaturated bond among them.

The divalent hydrocarbon group, which may have a substituent andcontains a 4 or more-carbon chain, includes a divalent hydrocarbon grouphaving a 4 or more-carbon chain and a divalent substituted hydrocarbongroup having a 4 or more-carbon chain. The divalent hydrocarbon grouphaving a 4 or more carbon chain includes alkylene, alkylene having anunsaturated bond, and a divalent aromatic group, which have an at least4-carbon chain.

Alkylene having a 4 or more-carbon chain includes a linear, branched, orcyclic alkylene having an at least 4-, preferably 4 to 20-, and morepreferably 4 to 15-carbon chain. Specific examples thereof includetetramethylene, pentamethylene, hexamethylene, heptamethylene,octamethylene, nonamethylene, decamethylene, and—(CH₂)_(m)-o-C₆H₁₀—(CH₂)_(n)— and the like, wherein m and n are eachindependently 0 or a natural number, and m+n≧2.

The alkylene that has a 4 or more-carbon chain and an unsaturated bondand the like includes an alkylene containing at least one unsaturatedbond such as a double bond and the like in the 4 or more-carbon chain.The alkylene that has a 4 or more-carbon chain and an unsaturated bondmay have an at least 4-, preferably 4 to 20-, and more preferably 4 to15-carbon chain, and it may be linear, branched, or cyclic. Specificexamples thereof include alkenylene such as butenylene, pentenylene, andhexenylene.

The divalent aromatic group having a 4 or more-carbon chain includes anat least 4-carbon chain-containing divalent aromatic group having an atleast 4-, preferably 4 to 20-, and more preferably 4 to 15-carbon chain.A specific example thereof includes —(CH₂)_(p)-o-C₆H₄—(CH₂)_(q)—,wherein p and q are each independently 0 or a natural number, and p+q≧2,and the like.

The divalent substituted hydrocarbon group containing a 4 or more-carbonchain includes a divalent substituted hydrocarbon group derived from adivalent hydrocarbon group containing a 4 or more-carbon chain bysubstituting at least 1 hydrogen atom thereof with the aforementionedsubstituent other than a hydroxy group.

When either R¹ and R² or R² and R³ are bonded to form a ring, the ringformed includes an aliphatic ring, an aromatic ring and the like. Thealiphatic ring includes a cyclobutane ring, a cyclopentane ring, acyclohexane ring, a cyclohexene ring and the like. The aromatic ringincludes a benzene ring.

Preferable examples of a hydroxy fatty acid include ricinoleic acid.11-hydroxypalmitic acid; lesquerolic acid, 10-hydroxystearic acid and10-hydroxypalmitic acid, and the lactone precursor hydroxy fatty acidrepresented by following formulae.

The aforementioned hydroxy fatty acid is preferably ricinoleic acid,11-hydroxypalmitic acid, lesquerolic acid, 10-hydroxystearic acid,10-hydroxypalmitic acid, or the like among them.

The hydroxy fatty acid derivative used in the present invention ispreferably one derived from the aforementioned hydroxy fatty acid.Preferable examples of such hydroxy fatty acid derivative include analkyl ester of hydroxy fatty acid, a glyceride of hydroxy fatty acid andthe like.

A specific example of the alkyl ester of hydroxy fatty acid, forexample, is represented by the general formula (3):

wherein R⁴ represents alkyl and R¹ to R³ are as defined above.

Alkyl represented by R⁴ may be linear or branched. The alkyl includes analkyl having at least one, preferably 1 to 10, more preferably 1 to 6,and further preferably 1 to 3 carbon atoms. Specific examples thereofinclude methyl, ethyl, n-propyl, 2-propyl and the like.

A preferable alkyl ester of hydroxy fatty acid in the present inventionis one having 1 to 3 carbon atoms in its alkyl ester portion (forexample, methyl ester, ethyl ester, and propyl ester etc.). Specificexamples thereof include ethyl 11-hydroxypalmitate, ethyl ricinoleate,ethyl lesquerolate, ethyl 10-hydroxystearate, ethyl 10-hydroxypalmitateand the like.

The glyceride of hydroxy fatty acid includes a monoglyceride of hydroxyfatty acid, a diglyceride of hydroxy fatty acid, and a triglyceride ofhydroxy fatty acid. A specific example thereof, for example, isrepresented by the general formula (4):

wherein R⁶ to R⁸ each independently represent a hydrogen atom or a grouprepresented by the general formula (6):

wherein R¹ to R³ are as defined above, provided that at least one of R⁶to R⁸ independently represents a group represented by the above generalformula (6).

The glyceride of hydroxy fatty acid represented by the above-mentionedgeneral formula (4) includes one obtained by condensation between aglycerin and the hydroxy fatty acid represented by the above-mentionedgeneral formula (2).

A preferable example of the glyceride of hydroxy fatty acid, in thepresent invention, includes castor oil and the like.

The hydrolysate of a hydroxy fatty acid derivative used in the presentinvention can be produced by hydrolyzing the aforementioned hydroxyfatty acid derivative. For example, such hydrolysate can be produced bychemical or enzymatic hydrolysis such as processing with a hydrolasesuch as lipase and the like, alkaline treatment, or high-pressure steamprocessing, of a glyceride or alkyl ester of hydroxy fatty acid. Thehydrolysate of a hydroxy fatty acid derivative includes, but is notlimited to, a castor oil hydrolysate and a hydrolysate of ethyl11-hydroxypalmitate.

The hydroxy fatty acid and the hydroxy fatty acid derivative used in thepresent invention may be an optically active (R) or (S) form, or aracemic modification. In the present invention, for example, when anoptically active hydroxy fatty acid represented by the general formula(2-1):

wherein * represents a chiral carbon atom and R¹ to R³ are as definedabove is used as the hydroxy fatty acid represented by the generalformula (2), the resulting a lactone is an optically active lactone.When R¹ is identical to R², a carbon atom to which R¹ and R² are boundis not a chiral carbon atom.

When an optically active hydroxy fatty acid derivative is used as ahydroxy fatty acid derivative, the resulting a lactone is also anoptically active lactone.

For example, the optically active lactone can be obtained with the useof the optically active alkyl ester of hydroxy fatty acid represented bythe general formula (3-1):

wherein R¹ to R⁴ and * are as defined above, as the alkyl ester of thehydroxy fatty acid represented by the above-mentioned general formula(3) or the optically active glyceride of hydroxy fatty acid representedby the general formula (4-1):

wherein R⁹ to R¹¹ each independently represent a hydrogen atom or agroup represented by the general formula (6-1):

wherein R¹ to R³ and * are as defined above, provided that at least oneof R⁹ to R¹¹ is a group represented by the general formula (6-1), as theglyceride of hydroxy fatty acid represented by the above-mentionedgeneral formula (4).

The optically active glyceride of an hydroxy fatty acid includes anoptically active monoglyceride of hydroxy fatty acid, an opticallyactive diglyceride of hydroxy fatty acid, an optically activetriglyceride of hydroxy fatty acid and the like.

Further, when a hydrolysate of a hydroxy fatty acid derivative is usedin the present invention, the same is true of the use of an opticallyactive hydroxy fatty acid derivative as a hydroxy fatty acid derivativeto be hydrolyzed.

More specifically, if the hydroxy group-binding carbon hasR-configuration, the resulting optically active a lactone is anR-lactone. If the hydroxy group-binding carbon has S-configuration, theresulting optically active lactone is an S-lactone.

A preferable example of the optically active hydroxy fatty acidrepresented by the general formula (2-1) is an optically active form ofa hydroxy fatty acid that was presented as the aforementioned preferableexample of the hydroxy fatty acid. Optically active ricinoleic acid,optically active 11-hydroxypalmitic acid, optically active lesquerolicacid, optically active 10-hydroxystearic acid, optically active10-hydroxypalmitic acid and the like are preferable among them.

Preferable examples of the optically active alkyl ester of hydroxy fattyacid represented by the general formula (3-1) include optically activeethyl 11-hydroxypalmitate, optically active ethyl ricinoleate, opticallyactive ethyl lesquerolate, optically active ethyl 10-hydroxystearate,and optically active ethyl 10-hydroxypalmitate and the like.

A preferable example of the optically active glyceride of hydroxy fattyacid represented by the general formula (4-1) includes, for example,optically active castor oil.

In order to produce γ-decalactone as a lactone by the production methodof the present invention, at least one member selected from the groupconsisting of, but is not limited to castor oil a castor oilhydrolysate, ricinoleic acid, and lesquerolic acid is preferably used asa hydroxy fatty acid, a hydroxy fatty acid derivative, or a hydrolysateof a hydroxy fatty acid derivative. When producing δ-decalactone by theproduction method of the present invention, use of, but are not limitedto, 11-hydroxypalmitic acid and/or ethyl 11-hydroxypalmitate, as ahydroxy fatty acid, a hydroxy fatty acid derivative, or a hydrolysate ofa hydroxy fatty acid derivative, is preferable.

The hydroxy fatty acid, the hydroxy fatty acid derivative, or thehydrolysate of a hydroxy fatty acid derivative used in the presentinvention may be a commercial product or may be extracted from naturalsubstances. Alternatively, it may be manufactured as appropriate. Inorder to obtain a natural lactone by the production method of thepresent invention, use of a hydroxy fatty acid, a hydroxy fatty acidderivative, or a hydrolysate of a hydroxy fatty acid derivative that wasobtained by process other than chemical synthesis is preferable. Forexample, 11-hydroxypalmitic acid can be preferably extracted from ajalap or sweet potato for the purpose of its use in producing naturalδ-decalactone. In order to obtain an optically active lactone by theproduction method of the present invention as mentioned above, use of anoptically active form of a hydroxy fatty acid, a hydroxy fatty acidderivative, or a hydrolysate of a hydroxy fatty acid derivative-ispreferable.

A culture medium that is used for producing a lactone in the presentinvention contains at least one selected from the group consisting ofthe hydroxy fatty acid, the hydroxy fatty acid derivative, and thehydrolysate of the hydroxy fatty acid derivative mentioned above inamounts of 10% (w/v) to 50% (w/v), and preferably 15% (w/v) to 25%(w/v), per one liter of the medium.

The medium that is used for producing a lactone in the present inventioncan be prepared by adding other conventional components for cell culture(e.g., a nitrogen source etc.) to at least one selected from the groupconsisting of the hydroxy fatty acid, the hydroxy fatty acid derivative,and the hydrolysate of the hydroxy fatty acid derivative as mentionedabove, if needed. Other components to be added to the medium include,but are not limited to, nitrogen sources such as yeast extract, urea,corn steep liquor, ammonium sulfate, and diammonium hydrogen phosphate;additional carbon sources such as malt extract, polypeptone, andsaccharides such as glucose; inorganic salts such as manganese sulfate,calcium chloride, ferric chloride, ferrous sulfate, ferric sulfate, zincsulfate, copper sulfate, magnesium sulfate, cobalt chloride, sodiummolybdate, boron, and potassium iodide; coenzymes such as flavinmononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamideadenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate(NADP), and coenzyme A (CoA); nucleotides such as adenosine triphosphate(ATP); vitamins such as L-carnitine; and sterilized water. Cofactorssuch as inorganic salts, coenzymes, and vitamins can further increasethe amount of a lactone precursor hydroxy fatty acid and/or a lactoneproduced, and the amounts thereof to be added may be usually very small.A person skilled in the art can easily determine such other componentsfor preparing a medium in which Candida sorbophila can grow, asappropriate.

(3) Production of an Optically Active Lactone Using Candida sorbophila

In the present invention, culturing of the Candida sorbophila asdescribed in (1) above in the medium for producing a lactone asdescribed in (2) above enables Candida sorbophila to produce a lactoneprecursor hydroxy fatty acid and/or a lactone in the medium.

The lactone precursor hydroxy fatty acid that was produced by Candidasorbophila can be converted into a lactone by lactonization thereof. Thelactone precursor hydroxy fatty acid can be lactonized by any methodknown to a person skilled in the art. For example, the lactonization canbe carried out by heating treatment under acidic conditions. Morespecifically, such processing can be carried out by heating a culturecontaining the lactone precursor hydroxy fatty acid under conditions ofpH 3 to 5 and 100° C. for 20 minutes and thereby lactonizing a lactoneprecursor hydroxy fatty acid in the culture product. The lactonizedcompound derived from the lactone precursor hydroxy fatty acid is thelactone according to the present invention. In the present invention,lactonization can be carried out without the use of a pH adjustor.

The lactone obtained by lactonization of a lactone precursor hydroxyfatty acid that is produced by the aforementioned process or the lactoneproduced by Candida sorbophila can be recovered and/or isolated andpurified by a method known to a person skilled in the art.

The term “lactone precursor hydroxy fatty acid” used herein refers to ahydroxy fatty acid that can be lactonized. The “lactone precursorhydroxy fatty acid” according to the present invention is, preferably, ahydroxy fatty acid which has hydroxy in the 4- or 5-position from thecarbon atom of carboxy and has at least 4, preferably 5 to 22, morepreferably 5 to 17, and further preferably 5 to 12 carbon atoms.

A specific example of the lactone precursor hydroxy fatty acid includesa hydroxy fatty acid represented by the general formula (5):

wherein R⁵ represents an optionally substituted divalent hydrocarbongroup having a 2 or 3-carbon chain; R¹ and R² are as defined above; andalternatively, R² and R⁵ may be bonded to form a ring.

R⁵, an optionally substituted divalent hydrocarbon group, include adivalent hydrocarbon group having a 2 or 3-carbon chain and a divalentsubstituted hydrocarbon group having a 2 or 3 carbon-chain.

The divalent hydrocarbon group having a 2 or 3-carbon chain includesalkylene having a 2 or 3-carbon chain of ethylene or trimethylene andalkenylene having a 2 or 3-carbon chain of ethenylene or propenylene.

The divalent substituted hydrocarbon group having a 2 or 3-carbon chainis a group derived from the aforementioned divalent hydrocarbon grouphaving a 2 or 3-carbon chain by substitution of at least 1 hydrogen atomwith the aforementioned substituent other than hydroxy. A specificexample of the divalent substituted hydrocarbon group having a 2 or3-carbon chain includes propylene and the like. When R² and R⁵ arebonded to form a ring, the resulting ring includes an aliphatic ring andan aromatic ring. The aliphatic ring includes a cyclobutane ring, acyclopentane ring, a cyclohexane ring, a cyclohexene ring and the like.The aromatic ring includes a benzene ring and the like.

Specific examples of the lactone precursor hydroxy fatty acid used inthe present invention represented by the general formula (5), wherein R⁵represents hydrocarbon having a 2-carbon chain, includeγ-hydroxydecanoic acid, a hydroxy fatty acid represented by thefollowing formulae and the like.

Specific examples of the lactone precursor hydroxy fatty acidrepresented by the general formula (5), wherein R⁵ is a hydrocarbongroup having a 3 carbon chain, include δ-hydroxydecanoic acid and thehydroxy fatty acid represented by following formulae.

When an optically active substance of a hydroxy fatty acid, of a hydroxyfatty acid derivative, or of a hydrolysate of a hydroxy fatty acidderivative is used in the method of the present invention, the resultinglactone precursor hydroxy fatty acid is the optically active onerepresented by the general formula (5-1):

wherein R¹, R², R⁵, and * are as defined above. In the presentinvention, an optically active lactone precursor hydroxy fatty acid ispreferably used as the lactone precursor hydroxy fatty acid.

The lactone obtained by the production method of the present inventionis represented by, for example, the general formula (1):

wherein ring A represents a lactone ring; R¹ and R² are as definedabove, in which ring A and R² may be bonded to form a ring.

The lactone ring represented by ring A in the general formula (1)includes a γ-lactone ring represented by the following formula (7) and aδ-lactone ring represented by the following formula (8). Ring A may alsohave a substituent.

Specific examples of the rings formed together between ring A and R²include rings represented by the following formulae.

Examples of the a lactone obtained by the production method of thepresent invention include, but are not limited to, a lactone having 4 ormore carbon atoms. Specific examples thereof include γ-decalactone,γ-valerolactone, γ-hexalactone, γ-heptalactone, γ-octalactone,γ-nonalactone, γ-undecalactone, γ-dodecalactone, γ-tridecalactone,γ-tetradecalactone, δ-decalactone, δ-hexalactone, δ-heptalactone,δ-octalactone, δ-nonalactone, δ-undecalactone, δ-dodecalactone,δ-tridecalactone, δ-tetradecalactone, angelica lactone, whisky lactone,γ-jasmolactone, jasmine lactone, lactone of cis-jasmone, methylγ-decalactone, jasmolactone, menthalactone, n-butyl phthalide,propylidene phthalide, butylidene phthalide,4,6,6,(4,4,6)-trimethyltetrahydropyran-2-one, δ-2-decenolactone,coumarin, dihydrocoumarin, cyclohexyl lactone, 6-methylcoumarin, and alactone represented by the following formulae.

In the above formulae, R represents a protecting group.

Among the aforementioned, a lactone having carbon numbers 5 to 12, suchas γ-decalactone, γ-valerolactone, γ-hexalactone, γ-heptalactone,γ-octalactone, γ-nonalactone, γ-undecalactone, γ-dodecalactone,γ-tridecalactone, γ-tetradecalactone, δ-decalactone, δ-heptalactone,δ-hexalactone, δ-octalactone, δ-nonalactone, δ-undecalactone,δ-dodecalactone, δ-tridecalactone, and δ-tetradecalactone, areparticularly preferable.

When an optically active form of a hydroxy fatty acid, a hydroxy fattyacid derivative, or a hydrolysate of a hydroxy fatty acid derivative isused in the production method of the present invention, the resultinglactone is an optically active lactone.

An example of the optically active lactone, for example, is representedby general formula (1-1):

wherein ring A, R¹, R², and * are as defined above.

The term “optically active lactone” used herein refers to a lactone thatis optically active substance (for example, an R or S form). Theoptically active lactone produced by the method of the present inventionis generated by lactonization of a lactone precursor hydroxy fatty acidthat is produced from a hydroxy fatty acid by the use of Candidasorbophila. Alternatively, the optically active lactone is produced froma hydroxy fatty acid by the use of Candida sorbophila.

The optically active lactone obtained by the production method of thepresent invention include, but are not limited to, an optically activelactone having at least 5 and preferably 5 to 12 carbon atoms. Forexample, an optically active form of the lactone presented as a specificexample above can be obtained. Preferable examples thereof include anoptically active γ-decalactone, an optically active γ-valerolactone, anoptically active γ-hexalactone, an optically active γ-heptalactone, anoptically active γ-octalactone, an optically active γ-nonalactone, anoptically active γ-undecalactone, an optically active γ-dodecalactone,an optically active γ-tridecalactone, an optically activeγ-tetradecalactone, an optically active 8-decalactone, an opticallyactive δ-hexalactone, an optically active δ-heptalactone, an opticallyactive δ-octalactone, an optically active δ-nonalactone, an opticallyactive δ-undecalactone, an optically active δ-dodecalactone, anoptically active δ-tridecalactone, and an optically activeδ-tetradecalactone.

(4) Production of an Optically Active γ-decalactone by the Method of thePresent Invention

The method for producing an optically active lactone of the presentinvention is hereafter described in greater detail by employing theproduction of an optically active γ-decalactone as an example. Themethod for producing an optically active lactone of the presentinvention can be basically carried out by procedures that are the sameas those for producing an optically active γ-decalactone.

a) Medium Used for Culture for Producing an Optically Activeγ-hydroxydecanoic Acid and/or an Optically Active γ-decalactone (Mediumfor Main Culture)

According to the present invention, at least one selected from the groupconsisting of castor oil, a castor oil hydrolysate, ricinoleic acid, andlesquerolic acid is used as a carbon source for the medium whenculturing for the purpose of producing an optically activeγ-hydroxydecanoic acid and/or an optically active γ-decalactone(referred to as the main culture in the Examples). In the presentinvention, a castor oil hydrolysate refers to a mixture obtained bychemically or enzymatically hydrolyzing castor oil. A castor oilhydrolysate includes a hydrolysate obtained by hydrolyzing castor oil bythe use of lipase (hereafter it is referred to as “lipase-treatedhydrolysate”). A main component of the hydrolysate obtained byhydrolyzing castor oil by the use of lipase is ricinoleic acid.Accordingly, a castor oil hydrolysate is mainly composed of, forexample, ricinoleic acid that is a main fatty acid consisting castoroil.

Any lipase can be used for hydrolyzing castor oil without particularlimitations as long as it can produce ricinoleic acid from castor oil.Examples of such lipase, for example, include: Lipase OF and Lipase MY(Meito Sangyo Co., Ltd.); and Newlase F3G, Lipase A “Amano” 6, Lipase AY“Amano” 30G, Lipase F-AP 15, Lipase G “Amano” 50, Lipase M “Amano” 10,and Lipase R “Amano” G (Amano Enzyme Inc.). A lipase-treated hydrolysatecan be obtained under conditions where a hydrolysate mainly composed ofricinoleic acid is generated from castor oil, for example, viaincubation with the addition of 0.5 g of lipase per 100 g of castor oilat 30° C. for 24 hours. This lipase-treated hydrolysate can be directlyused in mixture form.

When a castor oil hydrolysate is contained in the main culture mediumaccording to the present invention, a lipase-treated hydrolysate ispreferably added to the medium. In the production method of the presentinvention, however, inclusion of castor oil in the medium results in theinclusion of a lipase-treated hydrolysate in the medium after theculture due to the following reason. That is, according to the method ofthe present invention, a hydrolysate is generated from castor oil in amedium with the aid of the Candida sorbophila that is used in thepresent invention. Accordingly, addition of a lipase-treated hydrolysateis an optional step if castor oil is contained in the medium.

In the present invention, at least one selected from the groupconsisting of castor oil, a castor oil hydrolysate, ricinoleic acid, andlesquerolic acid may be used. These substances may be used alone, or inany combinations of two or more as appropriate. Among them, castor oiland/or a castor oil hydrolysate are particularly preferable.

The concentration of at least one selected from the group consisting ofcastor oil, a castor oil hydrolysate, ricinoleic acid, and lesquerolicacid to be added to the medium is generally about 10% to 50% (w/v), andpreferably about 15% to 25% (w/v) per liter of the medium.

The medium used in the present invention may further contain othercomponents such as yeast extract, malt extract, polypeptone, andglucose, if needed. Such medium containing additional components can beemployed as a nutrient medium to produce the γ-hydroxydecanoic acidand/or optically active γ-decalactone of the present invention.

Yeast extract, urea, corn steep liquor, ammonium sulfate, diammoniumhydrogen phosphate, and the like can be incorporated into theaforementioned medium as nitrogen sources. Also, malt extract,polypeptone, and saccharides such as glucose and the like can beincorporated into the medium as additional carbon sources.

Alternatively, a synthetic medium containing at least one selected fromthe group consisting of castor oil, a castor oil hydrolysate, ricinoleicacid, and lesquerolic acid as a single carbon source may also be used.This synthetic medium may further contain additional components such asthe additional nitrogen sources or carbon sources mentioned above.

The yield of an optically active γ-hydroxydecanoic acid and/or anoptically active γ-decalactone produced can be further increased byoptionally adding a variety of cofactors to the aforementioned medium.

Cofactors include: inorganic salts such as manganese sulfate, calciumchloride, ferric chloride, ferrous sulfate, ferric sulfate, zincsulfate, copper sulfate, magnesium sulfate, cobalt chloride, sodiummolybdate, boron, and potassium iodide; coenzymes such as flavinmononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamideadenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate(NADP), and coenzyme A (CoA); nucleotides such as adenosine triphosphate(ATP); and vitamins such as L-carnitine. The amount of a cofactor to beadded may be very small.

The main culture medium used in the present invention includes a mediumprepared by adding at least one selected from the group consisting ofcastor oil, a castor oil hydrolysate, ricinoleic acid, and lesquerolicacid to YM medium, potato dextrose medium, or potato sucrose medium anda medium prepared by adding any of the aforementioned additionalcomponents to the medium mentioned above.

b) Production of an Optically Active γ-hydroxydecanoic Acid and/or anOptically Active γ-decalactone by Main Culture

In the present invention, γ-hydroxydecanoic acid and/or an opticallyactive γ-decalactone can be produced and accumulated in a medium byculturing the Candida sorbophila according to the present invention inthe medium described in a) above.

The Candida sorbophila according to the present invention may bedirectly inoculated and cultured on the medium a). However, preferably,such Candida sorbophila is previously subjected to seed culture in aconventional medium, and the resultant is then inoculated or spread onthe medium a) for culture. Conditions for seed culture may be identicalto those for culture (main culture) for producing γ-hydroxydecanoic acidand/or an optically active γ-decalactone. However, the conditions arenot particularly limited as long as the Candida sorbophila strain can beproliferated. A conventional medium that is used for seed culture may besolid or liquid. It may or may not contain at least one selected fromthe group consisting of castor oil, a castor oil hydrolysate, ricinoleicacid, and lesquerolic acid. A seed culture medium includes YM slant agarmedium and potato dextrose slant agar medium. The amount of the seedculture product to be added to the medium for main culture is notparticularly limited. When the seed culture product is, for example,liquid, however, a culture product having an absorbance at 610 nm (OD610) of approximately 30 is preferably added in amounts of 1% to 3%relative to the amount of the main culture medium. Alternatively, seedculture may be followed by preculture, and main culture may be thencarried out in the medium a). Such two-phase culture is suitable formass-production and is particularly useful for industrial production.Conditions for preculture may be identical to those for main culture.However, the conditions are not particularly limited as long as theCandida sorbophila strain can be proliferated. The seed culture mediummay or may not contain at least one selected from the group consistingof castor oil, a castor oil hydrolysate, ricinoleic acid, andlesquerolic acid.

In the production method of the present invention, culture conditionsare aerobic. Culture temperature is selected as appropriate in the rangeof generally 20° C. to 35° C., and preferably 24° C. to 30° C. The pHlevel of the medium is selected as appropriate generally between 5 and7, and preferably between 5.5 and 6.5. Culture is carried out, forexample, in shaking culture in a shake flask or a fermenter (e.g., afermenter equipped with an agitator and aerator).

The duration of main culture is not particularly limited as long as itis long enough to produce an optically active γ-hydroxydecanoic acidand/or an optically active γ-decalactone. Preferably, the duration isselected in a manner such that the amount of optically activeγ-hydroxydecanoic acid and/or optically active γ-decalactone producedreaches the maximal level. Such duration varies depending on, forexample, the composition of the medium, the amount of at least oneselected from the group consisting of castor oil, a castor oilhydrolysate, ricinoleic acid, and lesquerolic acid to be added as asubstrate, and the aeration and agitation efficiency in accordance withthe culture apparatus employed. In the case of culture using a shakeflask as a culture apparatus, for example, the culture duration may beselected as appropriate in the range of generally 1 hour to 30 days, andpreferably 12 hours to 25 days. In the case of culture using afermenter, the culture duration may be selected as appropriate in therange of generally 1 day to 20 days, and preferably 3 to 10 days. Use ofa fermenter can result in the completion of culture within a relativelyshort period of time and thus is preferable from the viewpoint ofproduction efficiency.

The culture duration that is long enough to bring about the adequateamount of optically active γ-decalactone produced may be determined inthe following manner. The optically active γ-decalactone produced in amedium is sampled over the time course. Alternatively, theγ-hydroxydecanoic acid produced in a medium is sampled over the timecourse and then lactonized. The amount of the resulting optically activeγ-decalactone is then determined by gas chromatography (GC), thin-layerchromatography (TLC), or gas chromatography/mass spectrometry (GC-MS)and is further compared with the standard if needed.

c) Production of an Optically Active γ-decalactone From an OpticallyActive γ-hydroxydecanoic Acid

The optically active γ-hydroxydecanoic acid as produced in a medium inthe manner described above can be used as a pharmaceutical intermediateor the like in that state. In the present invention, however, theγ-hydroxydecanoic acid is further used to produce an optically activeγ-decalactone by conversion of the γ-hydroxydecanoic acid bylactonization.

Lactonization may be carried out by any conventional technique thereof.Lactonization of the optically active γ-hydroxydecanoic acid of thepresent invention may be carried out after the γ-hydroxydecanoic acidproduced in the aforementioned medium is recovered by a conventionaltechnique. Alternatively, it may be carried out while the culture mediumcontains the optically active γ-hydroxydecanoic acid by directlactonization of the medium after the completion of culture.

The aforementioned lactonization may be carried out by a conventionaltechnique, such as lactonization in a culture solution. A specificexample of such method is one wherein an acid such as dilutehydrochloric acid or dilute sulfuric acid is added to the culturesolution after the completion of culture to acidify the culturesolution.

In the present invention, lactonization is preferably carried outwithout acidifying the culture solution in order to obtain an opticallyactive γ-decalactone as a compound having properties as close aspossible to those thereof in a natural state. Since the pH level of theculture solution after the completion of culture is already in an acidicrange of between 3.0 and 4.5 in the present invention, an acidic culturesolution can be obtained without further acidifying the culturesolution. Lactonization of the optically active γ-hydroxydecanoic acidcontained in the culture solution can be realized for such acidicculture solution by heating it for generally approximately 10 minutes to1 hour, and preferably approximately 10 to 30 minutes, withoutacidifying the culture solution. This heating temperature is set atapproximately 70° C. to 130° C., and preferably at approximately 90° C.to 120° C. When lactonization is carried out in the present invention,the pH level of the medium within the acidic conditions is sufficient,and it is preferably between pH 2 and 5.

In the present invention, such lactonization of an optically activeγ-hydroxydecanoic acid can result in the conversion of the opticallyactive γ-hydroxydecanoic acid into the optically active γ-decalactone.

The obtained optically active γ-decalactone may be recovered andpurified from a culture solution by a conventional technique such assolvent extraction and distillation after cells are separated andremoved from the culture solution by centrifugation or other means.

According to the method for producing optically active γ-decalactone ofthe present invention, R-γ-decalactone of high optical purity can beobtained.

The lactone obtained by the production method of the present inventioncan be used for flavor and fragrance substances, pharmaceuticalintermediates, and the like. For example, R-γ-decalactone can be usedfor adding, strengthening, or enhancing organolepticities for beverages,chewing gum, fruit juice, tobacco products, pharmaceutical preparations,flavor and/or fragrance preparations, scented products, and the like.R-γ-decalactone has an aroma that is stronger than that ofS-γ-decalactone and advantageously has a more natural fruit-like aromaas its characteristic (A. Mosandle et al., J. Agric. Food Chem., 37,413, 1989).

(5) Conclusion

The production method of the present invention is characterized by useof Candida sorbophila. According to the method of the present invention,an optically active lactone such as R-γ-decalactone or R-δ-decalactonecan be obtained with high optical purity. Also, the method for producingoptically active 7-decalactone of the present invention has the highefficiency in terms of producing R-γ-decalactone. Such high productionefficiency provided by the method of the present invention is probablydue to the fact that presence of Candida sorbophila less leads todestroy the generated R-γ-decalactone in the production system of thepresent invention. Particularly, it is presumed that Candida sorbophiladoes not decompose R-γ-decalactone, resulting in the high productionefficiency. However, it is noted that the technical scope of the presentinvention should not be limited based on such hypothetical theory.

The method of the present invention facilitates the effective productionof an optically active lactone of high purity and can improve theworking efficiency in industrial production.

PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is hereafter described in more detail withreference to the examples, although the technical scope of the presentinvention is not limited thereto.

In the following examples, gas chromatography (GC) analysis was carriedout under the following conditions.

Apparatus: Model: 5890, Hewlett Packard

Column: BC-WAX (0.25 mm (diameter)×30 m (length), GL Sciences)

Internal standard sample: ethyl decanoate (1.0 v/v%)

The optical purities of the lactones produced in the Examples wereassayed via GC analysis (apparatus: G3000, column: Chirasil-DEX-CB, 0.25mm (diameter)×25 m (length), Hitachi).

EXAMPLE 1

The FC 58 strain was inoculated on YM slant agar medium and cultured at27° C. for 3 days for activation. A medium for preculture was preparedin the following manner. At the outset, 0.09 g of yeast extract, 0.09 gof malt extract, 0.15 g of polypeptone, and 0.3 g of glucose were placedin a 300-mL volume Erlenmeyer flask, distilled water was added theretoto bring the total volume to 30 mL, pH 6. The resultant was sterilizedin an autoclave at 121° C. for 15 minutes. The thus prepared medium wascooled, the FC 58 strain activated in the manner described above wasinoculated thereon, and shaking culture was carried out in a rotaryshake culture apparatus at 27° C. and 150 rpm for 24 hours forpreparation of a preculture solution. Further, a medium for thesubsequent main culture was prepared. Specifically, 0.3 g of yeastextract, 0.3 g of malt extract, and 0.5 g of polypeptone were placed ina 500-mL volume Sakaguchi flask, distilled water was added thereto tobring the total volume to 100 ml. pH 6. and 20 g of castor oil wasfurther added thereto. The resultant was sterilized in an autoclave at121° C. for 15 minutes. Subsequently, the medium for main culture wascooled, 2 ml of the aforementioned preculture solution was inoculatedthereon, and main culture was carried out via shaking culture at 27° C.and 150 rpm. The pH level was not adjusted during the culture. After theculture, the pH level of the culture solution was 4.06. Aseptic samplingof the culture solution in volumes of 5 ml each was initiated 3 daysafter the initiation of culture, sampling was conducted over the timecourse, and the sampled culture solutions (the samples) wereindependently heated to 100° C. for 20 minutes to lactonizeγ-hydroxydecanoic acid. After such lactonization, the sample wassubjected to extraction with ethyl acetate, and the separated organiclayer was quantified via gas chromatography (GC) analysis by theinternal standard method (with the use of ethyl decanoate as theinternal standard sample). As a result, the amount of R-γ-decalactoneproduced in the culture solution was found to become maximal 14 daysafter the initiation of culture. Such amount of R-γ-decalactone was 8.41g per liter of the main culture medium. The optical purity thereof was99% ee or higher.

EXAMPLE 2

R-γ-decalactone was produced in the same manner as in Example 1 exceptfor the use of 20 g of ricinoleic acid (purity of 80% or higher, WakoPure Chemical Industries, Ltd.) instead of 20 g of castor oil. As aresult, the amount of R-γ-decalactone produced in the culture solutionwas found to become maximal 20 days after the initiation of culture.Such amount of R-γ-decalactone was 21.89 g per liter of the main culturemedium. The optical purity thereof was 99% ee or higher.

EXAMPLE 3

R-γ-decalactone was produced in the same manner as in Example 2 exceptfor the addition of 1.7 mg of manganese sulfate (MnSO₄.H₂O), 0.55 mg ofcalcium chloride (CaCl₂.H₂O), 0.375 mg of ferric chloride (FeCl₃.H₂O),2.2 mg of zinc sulfate (ZnSO₄.H₂O), 0.4 mg of copper sulfate(CuSO₄.H₂O), 5.9 mg of magnesium sulfate (MgSO₄.H₂O), 0.28 mg of cobaltchloride (CoCl₂-H₂O), 0.26 mg of sodium molybdate (Na₂MoO₄.H₂O), 0.4 mgof boron (H₃BO₃), and 0.06 mg of potassium iodide (KI) to the mainculture medium. As a result, the amount of R-γ-decalactone produced inthe culture solution was found to become maximal 20 days after theinitiation of culture. Such amount of R-γ-decalactone was 27.37 g perliter of the main culture medium. The optical purity thereof was 99% eeor higher.

EXAMPLE 4

The FC 58 strain was inoculated on YM slant agar medium and cultured at27° C. for 3 days for activation. A medium for preculture was preparedin the following manner. At the outset, 0.3 g of yeast extract, 0.3 g ofmalt extract, 0.5 g of polypeptone, and 1.0 g of glucose were placed ina 500-mL volume Sakaguchi flask, distilled water was added thereto tobring the total volume to 100 ml, pH 6. The resultant was sterilized inan autoclave at 121° C. for 15 minutes. The thus-prepared medium wascooled, the FC 58 strain activated in the manner described above wasinoculated thereon, and shaking culture was carried out in a rotaryshake culture apparatus at 27° C. and 150 rpm for 24 hours forpreparation of a preculture solution. Further, a medium for thesubsequent main culture was prepared. Specifically, 6.0 g of yeastextract, 6.0 g of malt extract, 10.0 g of polypeptone, 34 mg ofmanganese sulfate (MnSO₄.H₂O), 11 mg of calcium chloride (CaCl₂.H₂O),7.5 mg of ferric chloride (FeCl₃.H₂O), 44 mg of zinc sulfate(ZnSO₄.H₂O), 8.0 mg of copper sulfate (CuSO₄.H₂O), 118 mg of magnesiumsulfate (MgSO₄.H₂O), 5.6 mg of cobalt chloride (CoCl₂.H₂O), 5.2 mg ofsodium molybdate (Na₂MoO₄.H₂O), 8.0 mg of boron (H₃BO₃), and 1.2 mg ofpotassium iodide (KI) were placed in a 5-L volume jar fermenter,distilled water was added thereto to bring the total volume to 2,000 ml,pH 6; and 400 g of ricinoleic acid (purity of 80% or higher, Wako PureChemical Industries, Ltd.) was further added thereto. The resultant wassterilized in an autoclave at 121° C. for 15 minutes. Subsequently, themedium for main culture was cooled, 40 ml of the aforementionedpreculture solution was inoculated thereon and main culture was carriedout under conditions of the agitation rate of 600 rpm, the aeration rateof 1000 ml/mm, and 27° C. The pH level was not adjusted during theculture. After the culture, the pH level of the culture solution was4.75. Aseptic sampling of the culture solution in volumes of 5 ml eachwas initiated 3 days after the initiation of culture, sampling wasconducted over the time course, and the sampled culture solutions (thesamples) were independently heated to 100° C. for 20 minutes tolactonize γ-hydroxydecanoic acid. After such lactonization, the samplewas subjected to extraction with ethyl acetate, and the separatedorganic layer was quantified by GC analysis by the internal standardmethod (with the use of ethyl decanoate as the internal standardsample). As a result, the amount of R-γ-decalactone produced in theculture solution was found to become maximal 10 days after theinitiation of culture. Such amount of R-γ-decalactone was 49.94 g perliter of the main culture medium. The optical purity thereof was 99% eeor higher.

EXAMPLE 5

R-γ-decalactone was produced in the same manner as in Example 4 exceptfor the use of a hydrolysate-obtained by treating 600 g of castor oilwith lipase (Lipase OF, Meito Sangyo Co., Ltd.) instead of 400 g ofricinoleic acid (purity of 80% or higher, Wako Pure Chemical Industries,Ltd.). As a result, the amount of R-γ-decalactone produced in theculture solution was found to become maximal 5 days after the initiationof culture. Such amount of R-γ-decalactone was 40.50 g per liter of themain culture medium. The optical purity thereof was 99% ee or higher.

EXAMPLE 6

Optically active γ-decalactone was produced in the same manner as inExample 2 except for the use of the Candida sorbophila strain ATCC74362, the Candida sorbophila strain ATCC 60130, or the Candidasorbophila strain IFO 1583 instead of the Candida sorbophila strain FC58. As a result, the amounts of γ-decalactone produced in the culturesolutions resulting from each of the aforementioned cell strains werefound to become maximal 19, 11, and 11 days after the initiation ofculture respectively. Such amounts of the γ-decalactone were 13.75 g,12.97 g, and 12.97 g per liter of the main culture medium, respectively.

EXAMPLE 7

R-γ-decalactone was produced in-the same manner as in Example 1, exceptthat the sampled culture solutions (samples) were not subjected tolactonization via heating. As a result, the amount of R-γ-decalactoneproduced in the culture solution was found to become maximal 14 daysafter the initiation of culture. Such amount of R-γ-decalactone was 8.41g per liter of the main culture medium. The optical purity thereof was99% ee or higher.

EXAMPLE 8

R-γ-decalactone was produced in the same manner as in Example 2, exceptthat the sampled culture solutions (samples) were not subjected tolactonization via heating. As a result, the amount of R-γ-decalactoneproduced in the culture solution was found to become maximal 20 daysafter the initiation of culture. Such amount of R-γ-decalactone was21.89 g per liter of the main culture medium. The optical purity thereofwas 99% ee or higher.

EXAMPLE 9

R-γ-decalactone was produced in the same manner as in Example 3, exceptthat the sampled culture solutions (samples) were not subjected tolactonization via heating. As a result, the amount of R-γ-decalactoneproduced in the culture solution was found to become maximal 20 daysafter the initiation of culture. Such amount of R-γ-decalactone was27.37 g per liter of the main culture medium. The optical purity thereofwas 99% ee or higher.

EXAMPLE 10

R-γ-decalactone was produced in the same manner as in Example 4. exceptthat the sampled culture solutions (samples) were not subjected tolactonization via heating. As a result, the amount of R-γ-decalactoneproduced in the culture solution was found to become maximal 10 daysafter the initiation of culture. Such amount of R-γ-decalactone was49.94 g per liter of the main culture medium. The optical purity thereofwas 99% ee or higher.

EXAMPLE 11

R-γ-decalactone was produced in the same manner as in Example 5, exceptthat the sampled culture solutions (samples) were not subjected tolactonization via heating. As, a result, the amount of R-γ-decalactoneproduced in the culture solution was found to become maximal 5 daysafter the initiation of culture. Such amount of R-γ-decalactone was40.50 g per liter of the main culture medium. The optical purity thereofwas 99% ee or higher.

EXAMPLE 12

R-γ-decalactone was produced in the same manner as in Example 6, exceptthat the sampled culture solutions (samples) were not subjected tolactonization via heating. As a result, the amounts of R-γ-decalactoneproduced in the culture solutions resulting from each of theaforementioned cell strains were found to become maximal 19, 11, and 11days after the initiation of culture, respectively. Such amounts ofR-γ-decalactone were 13.75 g, 12.97 g, and 12.97 g per liter of the mainculture medium, respectively.

EXAMPLE 13

The FC 58 strain was inoculated on YM slant agar medium-and cultured at27° C. for 3 days for activation. A medium for preculture was preparedin the following manner. At the outset, 0.09 g of yeast extract, 0.09 gof malt extract, 0.15 g of polypeptone, and 0.3 g of glucose were placedin a 300-ml volume Erlenmeyer flask, distilled water was added theretoto bring the total volume to 30 ml, pH 6. The resultant was sterilizedin an autoclave at 121° C. for 15 minutes. The thus prepared medium wascooled the FC 58 strain activated in the manner described above wasinoculated thereon, and shaking culture was carried out in a rotaryshake culture apparatus at 27° C. and 150 rpm for 24 hours forpreparation of a preculture solution.

Further, a medium for the subsequent main culture was prepared.Specifically, 0.09 g of yeast extract, 0.09 g of malt extract, 0.15 g ofpolypeptone, 0.51 mg of manganese sulfate (MnSO₄.H₂O), 0.17 mg ofcalcium chloride (CaCl₂.H₂O), 0.11 mg of ferric chloride (FeCl₃.H₂O),0.66 mg of zinc sulfate (ZnSO₄.H₂O), 0.12 mg of copper sulfate(CuSO₄.H₂O), 1.77 mg of magnesium sulfate (MgSO₄.H₂O), 0.08 mg of cobaltchloride (CoCl₂.H₂O), 0.08 mg of sodium molybdate (Na₂MoO₄.H₂O), 0.12 mgof boron (H₃BO₃), and 0.02 mg of potassium iodide (KI) were placed in a300-mL volume Sakaguchi flask, distilled water was added thereto tobring the total volume to 30 ml, pH 6, and 0.13 g of 11-hydroxy palmiticacid ethyl ester was further added thereto. The resultant was sterilizedin an autoclave at 121° C. for 15 minutes. Subsequently, the medium formain culture was cooled, 2 ml of the aforementioned preculture solutionwas inoculated thereon, and main culture was carried out in shakingculture at 27° C. and 150 rpm. The pH level was not adjusted during theculture. Aseptic sampling of the culture solution in volumes of 5 mleach was initiated 3 days after the initiation of culture, sampling wasconducted over the time course, and the sampled culture solutions (thesamples) were independently heated to 100° C. for 20 minutes tolactonize δ-hydroxydecanoic acid. After such lactonization, the samplewas subjected to extraction with ethyl acetate, and the separatedorganic layer was quantified by GC analysis by the internal standardmethod (with the use of ethyl decanoate as the internal standardsample).

As a result, the amount of S-δ-decalactone produced in the culturesolution was found to become maximal 11 days after the initiation ofculture. Such amount of S-δ-decalactone was 0.019 g per 30 ml of themain culture medium. The optical purity thereof was 96% ee or higher.

EXAMPLE 14

S-δ-decalactone was produced in the same manner as in Example 13, exceptthat the sampled culture solutions (samples) were not subjected tolactonization via heating. As a result, the amount of S-δ-decalactoneproduced in the culture solution was found to become maximal 11 daysafter the initiation of culture. Such amount of S-δ-decalactone was0.019 g per 30 ml of the main culture medium. The optical purity thereofwas 96% ee or higher.

REFERENCE EXAMPLE 1

γ-decalactone was produced in the same manner as in Example 2 except forthe use of the Yarrowia lipolytica strain IFO 0717 known to have highability of γ-decalactone production instead of the FC 58 strain. As aresult, the amount of γ-decalactone produced in the culture solution wasfound to become maximal 6 days after the initiation of culture. Suchamount of γ-decalactone was 4.90 g per liter of the main culture medium.Thereafter, the amount of γ-decalactone produced was monitored over thetime course. This revealed that the amount of γ-decalactone produced perliter of the main culture medium began to decrease 6 days after theinitiation of culture. The amount of γ-decalactone produced was 3.17 gper liter of the main culture medium 23 days after the initiation ofculture.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

Industrial Applicability

According to the present invention, a lactone, such as an opticallyactive lactone including an optically active γ-decalactone (e.g.,R-γ-decalactone) and an optically active δ-decalactone, can be easilyobtained with high production efficiency. Also, a lactone precursorhydroxy fatty acid, such as γ-hydroxydecanoic acid or δ-hydroxydecanoicacid, can be efficiently produced in an intermediary step while theproduction method of the present invention is carried out. In theproduction method of the present invention, the addition of anemulsifier or pH adjuster to a medium is not required, and carbonsources, for example, a hydroxy fatty acid, a hydroxy fatty acidderivative, and/or a-hydrolysate of a hydroxy fatty acid derivative, canbe added to the medium at concentrations that are high enough forlarge-scale production. Thus, the method of the present invention isvery useful in industrial production.

1. A method for producing a lactone comprising culturing Candidasorbophila in a medium containing at least one selected from a hydroxyfatty acid, a hydroxy fatty acid derivative, and a hydrolysate of ahydroxy fatty acid derivative, and recovering the produced lactone fromthe medium.
 2. A method for producing a lactone comprising culturingCandida sorbophila in a medium containing at least one selected from thegroup consisting of a hydroxy fatty acid derivative, and a hydrolysateof a hydroxy fatty acid derivative, and lactonizing a lactone precursorhydroxy fatty acid produced in the medium.
 3. The method according toclaim 1 or 2, wherein the Candida sorbophila is at least one selectedfrom Candida sorbophila strain ATCC 74362, Candida sorbophila strainATCC 60130, the Candida sorbophila strain IFO 1583, and the Candidasorbophila strain FC 58 deposited under the accession number FERMBP-8388.
 4. The method according to claim 1 or 2, wherein the lactone isrepresented by general formula (1):

wherein ring A represents a lactone ring; R¹ represents a hydrogen atom,a hydrocarbon group, a substituted hydrocarbon group, a heterocyclicgroup, or a substituted heterocyclic group; and R² represents a hydrogenatom, a hydrocarbon group, or a substituted hydrocarbon group; in whichring A and R² may be bonded to form a ring.
 5. The method according toclaim 1 or 2, wherein the lactone is an optically active lactone.
 6. Themethod according to claim 1 or 2, wherein the hydroxy fatty acid isrepresented by general formula (2):

wherein R¹ represents a hydrogen atom, a hydrocarbon group, asubstituted hydrocarbon group, a heterocyclic group, or a substitutedheterocyclic group; R² represents a hydrogen atom, a hydrocarbon group,or a substituted hydrocarbon group; and R³ represents an optionallysubstituted divalent hydrocarbon group having a 4 or more-carbon chain;in which R² and R³ may be bonded to form a ring.
 7. The method accordingto claim 1 or 2, wherein the hydroxy fatty acid derivative is an alkylester of hydroxy fatty acid or a glyceride of hydroxy fatty acid.
 8. Themethod according to claim 7, wherein the alkyl ester of hydroxy fattyacid is represented by general formula (3):

wherein R¹ represents a hydrogen atom, a hydrocarbon group, asubstituted hydrocarbon group, a heterocyclic group, or a substitutedheterocyclic group; R² represents a hydrogen atom, a hydrocarbon group,or a substituted hydrocarbon group; R³ represents an optionallysubstituted divalent hydrocarbon group having a 4 or more-carbon chain;and R⁴ represents an alkyl group; in which R² and R³ may be bonded toform a ring.
 9. The method according to claim 7, wherein the glycerideof hydroxy fatty acid is represented by general formula (4):

wherein R⁶ to R⁸ each independently represents a hydrogen atom or agroup represented by general formula (6):

wherein R¹ represents a hydrogen atom, a hydrocarbon group, asubstituted hydrocarbon group, a heterocyclic group, or a substitutedheterocyclic group; R² represents a hydrogen atom, a hydrocarbon group,or a substituted hydrocarbon group; R³ represents an optionallysubstituted divalent hydrocarbon group having a 4 or more-carbon chain;and R⁴ represents an alkyl group; in which R² and R³ may be bonded toform a ring, provided that at least one of R⁶ to R⁸ is a grouprepresented by the above general formula (6).
 10. The method accordingto claim 1 or 2, wherein Candida sorbophila is cultured in a mediumcontaining at least one selected from the group consisting of castoroil, a castor oil hydrolysate, ricinoleic acid, 11-hydroxypalmitic acid,lesquerolic acid, 10-hydroxystearic acid, 10-hydroxypalmitic acid, andethyl 11-hydroxypalmitate.
 11. The method according to claim 2, whereinthe lactone precursor hydroxy fatty acid is a hydroxy fatty acid of 4 ormore carbon atoms having a hydroxy group at position 4 or 5 thereof. 12.The method according to claim 1 or 2, wherein the lactone is any oneselected from the group consisting of γ-decalactone, γ-valerolactone,γ-hexalactone, γ-heptalactone, γ-octalactone, γ-nonalactone,γ-undecalactone, γ-dodecalactone, γ-tridecalactone, γ-tetradecalactone,δ-decalactone, δ-hexalactone, δ-heptalactone, δ-octalactone,δ-nonalactone, δ-undecalactone, δ-dodecalactone, δ-tridecalactone, andδ-tetradecalactone.
 13. A method for producing γ-dodecalactone precursorhydroxy fatty acid comprising culturing Candida sorbophila in a mediumcontaining at least one selected from castor oil, a castor oilhydrolysate, ricinoleic acid, and lesquerolic acid, and recovering theproduced γ-decalactone from the medium.
 14. A method for producingγ-decalactone comprising culturing Candida sorbophila in a mediumcontaining at least one selected from castor oil, a castor oilhydrolysate, ricinoleic acid, and lesquerolic acid, and recovering theproduced γ-decalactone from the medium.
 15. The method according toclaim 13 or 14, wherein γ-decalactone is an optically activeγ-decalactone.
 16. The method according to claim 13 or 14, wherein atleast one is castor oil and/or a castor oil hydrolysate.
 17. A methodfor producing δ-decalactone comprising culturing Candida sorbophila in amedium containing 11-hydroxypalmitic acid and/or ethyl11-hydroxypalmitate and recovering the produced δ-decalactone from themedium.
 18. A method for producing δ-decalactone comprising culturingCandida sorbophila in a medium containing 11-hydroxypalmitic acid and/orethyl 11-hydroxypalmitate and lactonizing δ-hydroxydecanoic acidproduced in the medium.
 19. The method according to claim 17 or 18,wherein δ-decalactone is an optically active δ-decalactone.
 20. Themethod according to claim 13, 14, 17, or 18, wherein the Candidasorbophila is at least one selected from the group consisting of theCandida sorbophila strain ATCC 74362, Candida sorbophila strain ATCC60130, the Candida sorbophila strain IFO 1583, and the Candidasorbophila strain FC 58 deposited under the accession number FERMBP-8388.